Hematopoietic stem cell transplant (HCT) is responsible for impressive cure rates of chemotherapy refractory leukemia and other malignancies. However, life-threatening opportunistic infections including cytomegalovirus (CMV) diminish full curative potential of HCT by raising morbidity and mortality throughout post-HCT recovery. Antiviral drugs which limit CMV viremia exact a cost of morbidity including renal dysfunction, neutropenia and immune suppression. Substituting toxic antivirals with a vaccine that stimulates multiple immune mechanisms may improve HCT outcomes. The vaccine is composed of an HLA promiscuous tetanus T-helper epitope covalently attached to an HLA-A2-restricted CTL epitope from CMV that stimulated a strong immune response in healthy adults and HCT recipients (HCT-R) when combined with a single stranded oligodeoxyonucleo-tide adjuvant and TLR9 agonist, PF03512676 (Pfizer). Subsequent to the completed Phase 1b trial in healthy adults, we initiated a randomized 2-arm pilot Phase 1b trial (Pilot) in both matched related (MRD) and unrelated (URD) donor HCT-R. Interim analysis of the Pilot in HCT-R supports the vaccine concept because preliminary data shows greater CMV-specific immunity, lower rates of CMV reactivation and chronic GVHD in the vaccine versus observational arm. In this application, we will advance this vaccine concept (CMVPepVax) by conducting 2 randomized, blinded and placebo-controlled Phase 2 trials to prevent CMV reactivation in HCT-R jointly with the University of Minnesota. In Specific Aim (SA) 1, we will conduct Trial 1 that is powered to test the primary endpoint of reduced CMV reactivation and disease in MRD-HCT. CMV-positive HCT-R will be randomized into a vaccine (VA) and a placebo (PA) arm, and given 2 injections spaced 4 weeks apart, while donors will be simultaneously and conjointly randomized to receive a single vaccination 2-5 weeks prior to stem cell collection. The effect of donor vaccination on improving HCT-R outcomes will be a secondary endpoint, thereby allowing up to 67% of donors to decline, yet still have power for effect on HCT-R outcomes. Immunologic 20 endpoints will be quantified by frequency measurements of CMV-specific T cells using HLA multimers and functional studies with T-box transcription factors, T-bet and Eomes. Importantly, this study will test our novel observations that NK cells respond to CMV reactivation after HCT by activating a specific NKG2C+ long-lasting (memory) response that can be mimicked using CMVPepVax, which would have significant general impact on the field. In SA2, Trial 2 will test protective function of CMVPepVax in higher risk URD and umbilical cord blood (UCB) recipients. This trial will employ the same format as Trial 1 except no donor involvement to address a broad range of URD settings, or lesser matched UCB grafts, all resulting in a higher risk of CMV reactivation and disease compared to MRD. The goal is to capitalize on our Phase 1 success with CMVPepVax by conducting Phase 2 studies that will not only establish the therapeutic benefit for HCT-R at risk for complications of CMV infection, but as well define the immunologic basis for this protection.